Multiple probe actuation

ABSTRACT

A method of actuating a plurality of probes. Each probe may be made of two or more materials with different thermal expansion coefficients which are arranged such that when the probe is illuminated by an actuation beam it deforms to move the probe relative to a sample. Energy is delivered to the probes by sequentially illuminating them with an actuation beam via an objective lens in a series of scan sequences. Two or more of the probes are illuminated by the actuation beam in each scan sequence and the actuation beam enters the objective lens at a different angle to an optical axis of the objective lens for each probe which is illuminated in a scan sequence. The actuation beam is controlled so that different amounts of energy are delivered to at least two of the probes by the actuation beam during at least one of the scan sequences.

FIELD OF THE INVENTION

The present invention relates to a method of actuating a plurality ofprobes, and apparatus operable to perform such a method.

BACKGROUND OF THE INVENTION

The speed of scanning in a probe microscope can be increased byoperating two or more cantilevers in parallel, such that data isacquired simultaneously from each probe. Parallel operation in scanningprobe microscopy (SPM) is a challenge because multiple probe detectionmust be implemented as well as independent actuation systems for eachcantilever. As a result, parallel SPM systems have in the past differedsignificantly from conventional SPM systems. For example, some systemshave deployed cantilevers with integrated piezo-resistive sensors, andintegrated zinc oxide Z-actuators (Quate et al Applied Physics Lettersvol 67 No 26 3918 (1995)). A major difficulty with such integratedsystems is the complexity and corresponding cost of the sensors. Thedesigns are also inflexible since changing a simple parameter such asthe pitch or spring constant of the cantilevers also requires a redesignof the layout and costly fabrication. As a result, parallel SPM systemsof this sort have not been widely used. There is therefore a need for aparallel probe microscope that is flexible in operation andconfiguration. Furthermore, such a system should incorporate a probedetection system and a probe actuation system that have at least theperformance of conventional SPM, while retaining compatibility withcantilever probes widely used in SPM.

Conventional probe microscopes employ piezo-electric elements to scanthe cantilever or specimen with nanometer level accuracy or better.However, such piezo-elements often have a limited speed of response dueto their size and mechanical characteristics. Smaller elements which canbe integrated into the cantilever can be employed for fast scanningapplications but the required fabrication and electrical connection is achallenge. Photothermal actuation has therefore been developed, in whichan infra-red laser is focused onto a cantilever and used to inducephotothermal bending of the cantilever for both z-actuation and resonantexcitation (Yamashita et al, Rev. Sci. Instrum. Vol 78, 083702 (2007).This approach is powerful and flexible, and can achieve a rapid responsetime due to the small size and short thermal time constant of themicromechanical cantilever. However this approach has not been used formultiple probe control due to the increased number of optical componentsneeded for alignment and focusing.

SUMMARY OF THE INVENTION

A first aspect of the invention provides a method of actuating aplurality of probes, the method comprising delivering energy to theprobes (optionally photothermal energy) so that the probes deformrelative to a sample (and optionally are heated), wherein the energy isdelivered to the probes by sequentially illuminating them with anactuation beam, optionally via an objective lens, in a series of scansequences. Two or more of the probes are illuminated by the actuationbeam in each scan sequence. Where an objective lens is provided, thenthe actuation beam enters the objective lens at a different angle to anoptical axis of the objective lens for each probe which is illuminatedin a scan sequence. The actuation beam is controlled so that differentamounts of energy are delivered to at least two of the probes by theactuation beam during at least one of the scan sequences.

A further aspect of the invention provides actuation apparatus foractuating a plurality of probes, the apparatus comprises a beam sourcefor generating an input beam; and an optical device arranged to receivethe input beam and transform the input beam to generate an actuationbeam which is output from the optical device at an output angle.Optionally an objective lens is provided. A controller is arranged tooperate the optical device to modulate the output angle of the actuationbeam over time so that the actuation beam is sequentially directed ontothe probes, optionally via the objective lens, thereby delivering energyto the probes so that the probes deform relative to a sample. The energyis delivered to the probes by sequentially illuminating them with theactuation beam, optionally via the objective lens, in a series of scansequences. Two or more of the probes are illuminated by the actuationbeam in each scan sequence. Where an objective lens is provided, thenthe actuation beam enters the objective lens at a different angle to anoptical axis of the objective lens for each probe which is illuminatedin a scan sequence. The controller is arranged to operate the opticaldevice and/or the beam source and/or an optical element in a path of theactuation beam or in a path of the input beam so that different amountsof energy are delivered to at least two of the probes by the actuationbeam during at least one of the scan sequences.

Instead of using multiple actuation beams, each generated by arespective laser, a single actuation beam is directed sequentially(typically one-by-one) onto the probes and its intensity, power and/orangle is controlled in order to independently modulate the positions ofthe probes relative to the sample.

The positions of one or more of the probes can thus be controlledindependently of other ones of the probes, for instance in order toindependently control each probe's separation from the sample forimaging purposes, or to move one or more of the probes into an imagingposition whilst leaving the rest of the probes in an inactive positionin which they do not interact with the sample.

The actuation beam is directed sequentially onto the probes, optionallyvia an objective lens, in a series of scan sequences. Each scan sequencemay illuminate the probes in the same order, or the order may changebetween sequences. All probes may be illuminated in all scan sequences,but more typically some of the probes are not illuminated in some of thescan sequences (for instance if they are not being used at the time).Typically each scan sequence occurs over a respective scan period, andtwo or more probes are illuminated by the actuation beam for arespective illumination period within each scan period. Eachillumination period may be continuous or may comprise a discontinuoustrain of illumination pulses. The actuation beam may remain turned on asit moves from probe to probe between illumination periods, or it may beturned off between the illumination periods.

Each probe may be illuminated at the same location on the probe everytime it is illuminated. Alternatively the location illuminated on agiven probe may vary between scan sequences. For instance each probe maybe illuminated at its base every other scan sequence (for instance thefirst, third and fifth sequence) and at its tip every other scansequence (for instance the second, fourth and sixth sequence).

The deformation of the probe may be a flexural deformation, a torsionaldeformation, or any other deformation. The deformation may be static ordynamic.

Typically the energy delivered to the probes is photothermal energy sothat the probes are heated and deform relative to the sample. In thiscase each probe may comprise two or more materials with differentthermal expansion coefficients which are arranged such that when theprobe is heated by an actuation beam it deforms to move the proberelative to a sample. Alternatively the probes may be made of a singlematerial—in this case they will deform due to a thermal gradientintroduced by heating a region of the probe and thus inducing mechanicalstress, typically between a side of the probe which is heated by theactuation beam and the opposite side of the probe. Alternatively theenergy may cause the probes to deform by some other mechanism such as byradiation pressure. Radiation pressure can be used in combination withhighly reflective probe coatings and ideally some form of cavity,possibly a mirror attached to the probe.

The actuation beam can be controlled in a number of ways. Typically theprobes are illuminated for a different amount of time and/or with adifferent power or intensity during at least one of the scan sequences.

As well as delivering different amounts of energy to different probesduring a single scan sequence, different amounts of energy are typicallyalso delivered to at least one of the probes by the actuation beamduring two or more scan sequences. This enables the position of a singleprobe to be changed from time to time independently of the other probes.

Typically each probe has a time constant, such as a thermal timeconstant, associated with the deformation which is longer than each ofthe scan sequences.

Typically the probes are sequentially illuminated by directing an inputbeam into an optical device; transforming the input beam to generate theactuation beam which is output from the optical device at an outputangle; and operating the optical device to modulate the output angle ofthe actuation beam over time so that the actuation beam is sequentiallydirected onto the probes, optionally via the objective lens. The opticaldevice typically comprises an acousto-optic or electro-optic modulatorwith a diffractive element which diffracts the input beam to generatethe output beam, and the output angle of the output beam is modulatedover time by applying an electrical or acoustic signal to thediffractive element, the signal modulating a refractive index of thediffractive element. Alternatively the optical device may comprise amirror which reflects the input beam and is rotated to modulate theoutput angle of the actuation beam over time. Typically the differentamounts of energy are delivered to at least two of the probes bycontrolling the input beam, by controlling the optical device, or bycontrolling the actuation beam with an optical element other than theoptical device in a path of the actuation beam.

The method may further comprise: directing a detection input beam intoan optical device; transforming the detection input beam with theoptical device into a plurality of output beamlets (which are optionallynot parallel with each other); splitting each output beamlet into asensing beamlet and an associated reference beamlet; simultaneouslydirecting each of the sensing beamlets onto an associated one of theprobes, optionally with the objective lens, to generate a reflectedbeamlet; combining each reflected beamlet with its associated referencebeamlet to generate an interferogram; and measuring each interferogramto determine the position of an associated one of the probes.

Typically the plurality of probes comprises ten or more probes.Optionally the plurality of probes may comprise one hundred or moreprobes.

The probes may be arranged in a single straight line, or in atwo-dimensional array.

The probes may be used in a number of applications, including (but notlimited to): scanning probe microscopy, for example for extremeultraviolet (EUV) mask inspection and review; biosensing to detectmultiple biomarkers; nanolithography, such as dip pen nanolithography inwhich scanning probes deposit chemical compounds on a substrate; or datastorage in which each probe has a heater allowing its temperature to beindependently raised to melt a polymer substrate followed by animprinting action by the probe producing a dent representing a binarydigit.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the invention will now be described with reference to theaccompanying drawings, in which:

FIG. 1 is a schematic drawing of a scanning probe microscope;

FIG. 2 illustrates the interferometer in detail;

FIGS. 3 and 4 show two alternative photodiode arrays;

FIG. 5 shows a linear array of seven probes and the illumination spotscreated by their associated sensing and actuation beamlets;

FIG. 6a shows a saw-tooth scanning pattern for the actuation beam;

FIGS. 6b-d show three different ways of modulating the intensity of theactuation beam to control the probes;

FIG. 7 shows an alternative scanning pattern which can be used tocontrol the probes without varying the intensity of the input beam;

FIGS. 8a-d show an example of how the deformation of two of the probescan be controlled as they traverse steps on the sample surface;

FIG. 9 is a schematic drawing of a biosensor; and

FIG. 10 shows an embodiment in which different objective lenses are usedfor the sensing beamlets and the actuation beam.

DETAILED DESCRIPTION OF EMBODIMENT(S)

With reference to FIG. 1, a scanning probe microscope that incorporatesan interferometer based sensing system and photothermal actuation systemin accordance with the present invention is shown. The microscopecomprises a moveable stage 1 adapted to receive a sample 1 a whosesurface is to be investigated by an array of thermal actuated bimorphprobes, only one of which is shown in FIG. 1. The scanning capability isprovided by two conventional drive systems: an x,y scanner 2 operable byan SPM controller 3 to provide relative motion of the probe array in theplane (x, y) of the sample 1 a; and a z positioning system comprisingpiezoelectric drivers 4 operable to move the probe and sample towardsand away from each other (z direction) over ranges larger than thatachievable by the photothermal actuation of the probe array.

The probe array comprises a linear array of cantilever beams 5 a, eachcarrying a tip 6 a which tapers to a point, and which is located towardsa distal end of the cantilever beam. The other (base or proximal) end ofeach cantilever beam 5 a is supported by a mount 7. In this embodiment,the z-positioning system 4 is connected to the probe mount 7.Alternatively, it may be connected to the sample stage 1.

The probe tip 6 a comprises a three dimensional, often conical orpyramidal structure that is located at the free end of each cantileverbeam 5 a. The tip tapers to a point that is its closest point ofinteraction with a surface under interrogation. The cantilever is thebeam itself, excluding the tip, that supports the tip at one end and atthe other is held by the microscope apparatus. The cantilever 5 a andtip 6 a together are referred to as the probe.

Each probe is generally fabricated from silicon or silicon nitride.Typically, the cantilever 5 a is around 50-200 μm long, 20-50 μm wideand around 0.2-2 μm thick, but this size can of course be variedaccording to application. The shape may also be varied: typically it isrectangular or triangular with, in the latter case, the tip in thevicinity of its apex. The tip is typically 5 μm at its base, 3-10 μmhigh and with an end radius of curvature of 2-20 nm. In use, the finepoint at the end of the tip 6 a is oriented towards the sample 1 a.Recently, smaller dimension probes have been fabricated for use atfaster imaging speeds. These probes have cantilevers around 5-20 μm longand 3-10 μm wide, with a correspondingly smaller tip. The tip may beformed as part of the cantilever beam fabrication process or added as apost processing step, for example, using electron beam deposition (EBD)to create a diamond like carbon (DLC) spike. Additionally, thecantilever beam is coated in a metal, typically, gold or aluminum, toincrease the reflectivity of the cantilever beam when using an opticalmethod to detection method.

The system is in principle capable of any conventional SPM imaging mode,and also more advanced modes developed for industrial inspection, suchas, the inspection of semiconductor wafers or photo-masks. The systemuses SLM units to create, steer and modulate multiple beams within theinterferometer sensing system and the photothermal actuation system,thereby allowing parallel operation of an array of cantilever probes.

With reference to both FIGS. 1 and 2, a detection laser 10 generates adetection input beam 11 which is incident on a Spatial Light Modulator(SLM) 12 where the beam is split into the required number of beamlets 13a-c. Typically the beam 11 is split into one beamlet 13 a-c for everycantilever in the array. Alternatively the beam 11 may be split into twoor more beamlets 13 a-c per cantilever to measure heights or relativeheights at different locations (for example one at the tip and anotherat the base to measure bending of the cantilever).

It would be practically difficult and complex to implement passiveoptical elements for this purpose, and so the SLM 12 is employed for theflexibility and ease of integration into an optical system whileallowing computer control for rapid alignment. A brief description ofthe principles of the SLM 12 follows. Suitable SLMs are supplied byBoulder Nonlinear Systems, Colorado, USA such as their XY Seriesproducts and Hamamatsu such as their X10468 Series products.

In an exemplary SLM, in order to modulate the phase of incident light, anematic liquid crystal SLM is aligned in a planar conformation. Here theliquid crystal director (i.e. long axis of the molecules) is orientedparallel to the polarization of the incident light. Upon application ofa voltage, the molecules tilt in a direction parallel with the directionof propagation of the optical field. This causes the incident light toencounter a reduced refractive index. The change in refractive indextranslates directly to a change in the optical path, and consequently aphase shift for the incident light. If enough voltage is applied, thevariation in refractive index ranges from the extraordinary index (forno applied voltage) to the ordinary index (maximum tilt of themolecules). A typical change in the refractive index for maximum appliedvoltage is 0.2. In the preferred embodiment the SLM 12 uses very largescale integration (VLSI) to address an array of liquid crystalmodulators. The VLSI addressing allows for multiplexing to achieveindividually addressable pixels across the entire optical aperture. Thisflexibility results in a randomly addressable phase mask that acts as anoptical phased array with the potential for phase correction. The SLMoptical head itself consists of a layer of liquid crystal sandwichedbetween a cover glass and a VLSI backplane.

The beamlets 13 a-c then enter an interferometer 14, shown in detail inFIG. 2, where they are split by a beam splitter 20 into sensing beamlets15 a-c and reference beamlets 16 a-c. The beam splitter 20 may create alateral shift of the beam but no angular deviation.

The sensing beamlets 15 a-c leave the interferometer and are reflectedby a fixed mirror 16 onto a tracking mirror 17 (shown in FIG. 1 butomitted from FIG. 2) that steers the beamlets during XY scanning so thatthey remain optimally positioned on the cantilevers. The tracking mirror17 comprises a scanning mirror which reflects the beamlets 15 a-c at anangle which varies synchronously with the x,y scanner 2. Alternativelythe tracking mirror 17 may be omitted and this steering functionundertaken by the SLM 12, depending on the speed requirements. Thesensing beamlets 15 a-c, having been steered by the tracking mirror 17or SLM 12, are then focused by an objective lens 18 onto the ends of thecantilevers where they are reflected back towards the objective lens 18.The lens 18 collects and collimates the reflected beamlets 19 a-c andprojects them back into the interferometer 14, where they are split intotwo components 30 a-c and 31 a-c by a phase shifting beamsplitter 21 andincident on photodiodes 22,23. The reference beamlets 16 a-c are eachsplit by the phase shifting beamsplitter 21 into two components 32 a-cand 33 a-c and incident on the photodiodes 22,23 they are interferedwith their associated reference beamlets 30 a-c and 31 a-c. The coatingof the phase shifting beam splitter generates a phase quadraturerelationship between the pair of interferogram beams produced by theoverlapping reference beamlets 32 a-c and 33 a-c and associatedreference beamlets 30 a-c and 31 a-c.

Although the lens 18 is illustrated as a single lens element, it will beunderstood that it may comprise an assembly of multiple lens elements.

After signal processing the signals are sent from the interferometer 14to the SPM controller 3, which is adapted for parallel operation, eachdata channel representing the position of a point on a cantilever withinthe array with respect to a reference point.

The reference beamlets 16 a-c are directed towards a suitably positionedretro-reflector 24, and thereafter to the beam splitter 21, where thereference beamlets are split and recombined with the two sensing beamletcomponents to create first and second interferograms with a relativephase shift of 90 degrees. The interferograms are detected respectivelyby the first and second photodiodes 22,23. Interferometric methods ofextracting the path difference between two coherent beams in such ahomodyne interferometer are well known in the art and so will not bedescribed here.

The two interferograms should ideally produce signals from thephotodiodes which are complementary sine and cosine signals with a phasedifference of 90 degrees. The signals should have equal amplitudes withno DC offset and only depend on the displacement of the cantilever andwavelength of the laser. Due to practical limitations, such as imperfectoptical components and alignment, the signals are typically notperfectly harmonic, with equal amplitude, in phase quadrature andwithout a DC offset. Thus known methods can be used to monitor thephotodiode output signals while changing the optical path lengthdifference in order to determine and to apply corrections for theseerrors.

The phase quadrature signals from the photodiodes are suitable for usewith conventional interferometer reversible fringe counting and fringesubdividing techniques which, for example, may be implemented usingdedicated hardware, programmable hardware such as an FPGA or as aprogrammed computer. Methods for subdividing or interpolating based onthe arc tangent of the quadrature signals are well known and can providesub nanometer resolution.

Note that optionally the retro-reflector 24 may be replaced by a lensand a planar mirror. This might be advantageous in anon-infinity-corrected system where the probes are not at the focalplane of the objective lens 18.

The beamlets 13 a-c are steered by the SLM 12 so that the sensingbeamlets 15 a-c propagate at different angles relative to the opticalaxis 25 of the objective lens 18, such that when they reach theobjective lens 18 it focuses each beamlet at the required place on theback of each cantilever in the array, in the focal plane of the lens foran infinity-corrected system. The SLM 12 achieves this diffractive beamsteering with an optical phased array analogous to a radar system. Notethat the angular deviations of the beamlets from the axes of theinterferometer are only a few degrees at most, hence they areexaggerated in FIG. 2. The separation of the beamlets by the objectivelens 18 onto the cantilever array has correspondingly been greatlyexaggerated so that they can be visualized. Each beamlet is thenreflected back off the cantilever and collected by the objective lens18. The beamlet is then collimated by the objective and propagates backtowards the beam splitter 21, retaining however the same magnitude ofangular orientation with respect to the optic axis of the system.Meanwhile each reference beamlet has passed through the retroreflector24 and beam splitter 21 and the matching reference beamlets areoverlapped with the sensing beamlets as they propagate towards lenses26,27 in front of multi-segment photodiodes 22 and 23. These lenses26,27 focus the collimated beamlets down to a series of spots on themulti-segment photodiodes 22,23, each corresponding to a cantilever inthe array. The position of each spot is directly related to the angulardeviation of the beam from the optical axis, and hence the reference andsensing beamlets recombine to produce an intensity signal that can berelated to the optical path length between them. In this way, parallelinterferometric position sensing of each cantilever in the array can beachieved with sub-nanometer resolution and high bandwidth. Theinterferometer described herein is one example of a homodyne system. Theparticular system described offers a number of advantages to thisapplication. The use of two phase quadrature interferograms enables themeasurement of cantilever displacement over multiple fringes, and henceover a large displacement range. The use of a phase-shifting coating onthe beamsplitter 21 used to generate the pair of phase quadratureinterferograms reduces the interferometer insensitivity to polarisationeffects, for example arising from changes in polarisation as the lightbeam is reflected from the cantilever. Examples of an interferometerbased on these principles are described in U.S. Pat. No. 6,678,056 andWO2010/067129. Alternative interferometer systems capable of measuring achange in optical path length may also be employed with this invention,for example, a homodyne interferometer could be implemented usingpolarization methods to generate the two phase quadrature interferogramsor a heterodyne interferometer implemented by using a dual frequencylaser. A suitable homodyne polarisation based interferometer isdescribed in EP 1 892 727 and a suitable heterodyne interferometer isdescribed in U.S. Pat. No. 5,144,150 which could be adapted for use withthis invention.

The position of the probe will affect the path of the reflected beamlets19 a-c and position of their associated spots on the photodiode 22,23.Although the angle of the probe will affect the reflected angle of thebeamlet it is the height and position of the probe in the focal plane ofthe objective lens 18 which is particularly critical. In theory, lightpropagating from any angle from a single point in the focal plane of theobjective lens 18 will arrive at a single point on the photodiode 22,23.This is because the focusing lens 26,27 in front of the photodiode isforming an image of the object, i.e. probe array, placed in the focalplane of the objective lens 18. One way to visualise this is to considerthat light propagating from any angle from a single point in the focalplane of the objective lens 18 will produce a collimated beam of lightafter the objective lens which will be focused to a single point by thephotodiode focusing lens 26,27. Note, this is for an infinity correctedoptical system where the probe and photodiodes 22,23 are in the focalplane of the objective lens 18 and focusing lens 26,27 respectively.

Now considering a change in height of the probe, this will affect theformation of the spot on the photodiode. In fact, it will not onlyaffect the position of the spot but also the shape of the spot, i.e. itwill be out of focus. This will affect the ability to create aninterferogram with the reference beam. This can be visualised by thereflected beam of light after the objective being slightly converging ordiverging and thus the wave front of the beam will be distorted comparedto the reference beam which will affect the contrast of theinterferometer.

However it is not necessary for the sensing beamlets and referencebeamlets to completely overlap on the photodiode 22,23. FIG. 3 showspart of the photodiode 22, along with the overlapping spots associatedwith the sensing beamlets 30 a-c and reference beamlets 33 a-c. Theregion where the beams overlap will form an interferogram and the largerthe overlap the greater the intensity change of the constructive anddestructive interference associated with a path length change betweenthe sensing and reference beam lets. The regions which do not overlapsimply form a DC offset on the intensity measured by the photodiode. Ifthe degree of overlap between the spots changes, the magnitude of theintensity variation due to the interference will change, but the averageDC offset measured will remain the same. Obviously it is preferred tomaximise the overlap of the spots and thus signal to noise ratio,however, variations in the overlap can be accommodated.

The photodiode 22 has a body 40 and three photosensitive regions 41 a-c.The optical system is configured such as to direct each overlapping pairof beamlets onto the centre of a respective one of the photosensitiveregions 41 a-c, so that the output of each region 41 a-c represents thetrue instantaneous height of an associated one of the probes in the zdirection. This is independent of the position of the base of the proberelative to the tip i.e. of the deflection.

In the case of FIG. 3 the intensity of each interferogram is monitoredindependently as each interferogram is located on a separate region 41a-c of the photodiode 22. The pitch and position of the interferogramsis controlled by the magnification of the optical system which isdetermined by the focal length of the photodiode lens 26,27 divided bythe focal length of the objective lens 18.

Optionally a greater number of photodiode regions than interferogramscan be used as shown in FIG. 4 in which the photodiode 22 has nineregions 42 a-i and the regions over which each interferogram falls canbe summed to determine the interferogram's intensity. This allows foradjustments in pitch of the interferograms which may be required toaccommodate changes in probe pitch.

Note that it is possible to use a 2D photodiode array if a 2D array oflevers and thus interferograms is to be measured.

Returning to FIG. 1, the microscope has a photothermal sequentialactuation system for bending the probes which will now be described.Light from an IR laser 50 is incident on an acoustic-optic modulator(AOM) 51 comprising a transparent crystal 51 a (typically telluriumdioxide (TeO₂), crystalline quartz, or fused silica), a piezoelectrictransducer 51 b attached to the crystal 51 a, and an absorber 51 c. Thetransducer 51 b is used to excite a sound wave in the crystal 51 a witha frequency of the order of 100 MHz. An input beam 53 from the laser 50then experiences Bragg diffraction at the traveling periodic refractiveindex grating generated by the sound wave. The scattered actuation beam52 has a slightly modified optical frequency (increased or decreased bythe frequency of the sound wave) and a slightly different direction. Thechange in direction is smaller than shown in FIG. 1, because the wavenumber of the sound wave is very small compared with that of the inputbeam 53. The frequency and direction of the scattered actuation beam 53can be controlled via the frequency of the sound wave, whereas theacoustic power is the control for the optical powers. For sufficientlyhigh acoustic power, more than 50% of the optical power can bediffracted—in extreme cases, even more than 95%. The acoustic wave isabsorbed at the other end of the crystal by the absorber 51 c.

Within the AOM 51 the acoustic wave is provided by a radio frequency(RF) signal 58 to the AOM, controlled by an AOM controller (not shown).This AOM controller has three components, a Voltage ControlledOscillator (VCO); a Voltage Variable Attenuator (VVA); and an amplifier.The VCO provides a RF sine-wave output, the frequency of the RF outputis determined by an applied control voltage, and varies approximatelylinearly with it. The VVA attenuates the output from the VCO, the degreeof attenuation is controlled by varying the applied control voltage tothe VVA. The amplifier amplifies the output of the VVA, such that the RFoutput is sufficient to drive the AOM. The response of the AOM varieswith the frequency and amplitude of the input RF signal.

The actuation beam 52 is reflected off a mirror 80 which reflects thewavelength of the beam 52 but transmits other wavelengths. The beam 52then passes to the tracking mirror 17 (shared with the interferometer14) which maintains it in position on the cantilevers as they arescanned in XY. The actuation beam 52 is then focused by the objectivelens 18 onto the cantilevers and precise positioning is achieved bysteering the beam 52 sequentially onto the cantilevers using the AOM 51.The photothermal actuation system is capable of controlling thecantilevers in a number of ways, with great flexibility and control. Forexample, by steering the actuation beam 52 to the base of thecantilevers, the photothermal effect can be used to deflect thecantilever up and down for the purposes of cantilever selection, oralternatively, z-actuation in the case of conventional SPM feedbackcontrol.

FIG. 5 is a view showing a linear array of seven cantilevers 5 a-f eachilluminated by a respective sensing beamlet 15 a-g at its distal endabove the tip 6 a (which is on the opposite side of the cantilever andhence not shown in FIG. 5). One of the cantilevers is also illuminatedby the actuation beam 52 at its proximal end near the mount 7.

With a suitable objective lens 18 it is possible to achieve a spot sizefor the actuation beam 52 and sensing beamlets 15 a-g of only a fewmicrons, allowing the precise application of infra-red radiation to aspecific location on the cantilever as required for efficientphotothermal actuation. Using the SLM 12 it is also possible to controlthe size of the focused spot produced by each beamlet as shown in FIG. 6in which the actuation beam 52 creates a spot which is larger than thosecreated by the sensing beamlets 15 a-g.

The cantilevers 5 a-g are thermal biomorph structures, the materials ofwhich undergo differential expansion when heated. That is, they arecomposed of two (or more) materials, with differing thermal expansions.Typically, this will be a silicon or silicon nitride base with a gold oraluminium coating. The coating extends the length of the cantilever andcovers the reverse side from the tip. The actuation light source 50preferably emits light 53 of one or more wavelengths at which there is amaximum or peak in the absorption spectrum for the particular coating.For example the wavelength may be around the aluminium absorption peakat ˜810 nm. Other coating/wavelength combinations can be used, forexample gold has a higher absorption below 500 nm light. When this lightis incident on the coating side of the cantilevers, the aluminiumexpands to a greater degree than the silicon nitride, bending thecantilever such that the tip moves downwards, towards the sample. Ifillumination intensity is increased, the tip therefore moves closer tothe sample surface. Conversely, if the intensity is lowered, bending isdecreased and the tips are moved away from the sample. Otherarrangements of coating and base materials may result in bending in anopposite direction in response to illumination.

The cantilevers 5 a-g are shown with a constant spacing (pitch) betweenthem, but optionally the spacing between adjacent cantilevers may varyacross the width of the array.

Referring to FIG. 1: the actuation light source 50 and AOM 51 arecontrolled by the SPM controller 3 which controls the intensity of thelight 53 emitted from the actuation light source 50 and thus controlsthe intensity of the actuation beam 52 at a high modulation rate oftypically 100's of kHz to 10's of MHz. The intensity of the beam 52determines the degree of bend exhibited by the thermal bimorph probes(regardless of their material specifics) and so governs the tip—sampleseparation distance during the course of a scan.

Alternatively, the intensity of the actuation beam 52 may be modulatedby the AOM 51, by an optical element 70 in the path of the actuationbeam 52, or by an optical element 71 in the path of the input beam 53.The optical element 70, 71 could be for example an acousto-opticmodulator (AOM) or an electro-optic modulator (EOM).

The intensity of the actuation beam 52 is modulated as the scanprogresses in accordance with parameters that will now be described withreference to FIGS. 6a -d.

FIGS. 6a-d show how the AOM 51 and laser 50 are operated toindependently actuate each probe. The AOM 51 sequentially directs theactuation beam onto the probes in a series of scan sequences, six ofwhich are illustrated in FIG. 6a . FIG. 6a shows the variation over timeof the angle of the actuation beam with respect to the optical axis ofthe objective lens 2. The angle increases uniformly within each scanperiod from a minimum angle to a maximum angle, then returns to theminimum angle for the next scan period. Each probe is illuminated by theactuation beam for a respective part of each scan period.

FIG. 6b shows how the power or intensity of the actuation beam ismodulated to independently actuate only two probes. However it should benoted that the principles illustrated in FIG. 6b can be extended to anynumber of probes. A first one of the probes is illuminated during afirst illumination period T₁ to T₂ within each scan sequence, and asecond one of the probes is illuminated during a second illuminationperiod T₃ to T₄ within each scan sequence. The intensity of the inputbeam 53 (and hence the intensity of the actuation beam 52) is modulatedas shown by the dashed line in FIG. 6b , and the area of thecross-hatched rectangles show the amount of photothermal energy beingdelivered to each probe by the actuation beam during a scan sequence.Note that in this example the actuation beam is turned on at all timesas it traverses across a probe, and is also turned on as it traversesbetween the probes. The intensity of the actuation beam is modulated sothat the two probes are heated by the actuation beam by differentamounts during each of the scan sequences shown in FIG. 6b Also each oneof the probes is heated by the actuation beam by different amountsduring at least two of the scan periods. In this example each probe isheated by a gradually increasing amount during the first three scanperiods, then the second probe is no longer heated so it retracts whilethe first probe remains being heated for two further scan periods (sothe second probe continues moving towards the sample as the first proberetracts).

The scan period (shown in FIGS. 6a and 6b ) is of the order of 1microsecond The illumination period for each probe (T2-T1 and T3-T4) isof the order of 0.1 microseconds. Each probe has a thermal time constantassociated with the deformation which is longer than the scan period,the thermal time constant being typically of the order of 5-10microseconds or longer depending on the dimensions and geometry of thecantilever. This ensures that each probe does not cool down to asignificant extent during the time between each successive illuminationperiod for that probe. The thermal response of the cantilever can beapproximated as an exponential function where the response due toheating is:Deflection=constant*(1−e^(−time/thermal time constant)and the response due to cooling is:Defection=constant*e^(−time/thermal time constant).

The thermal time constant may vary slightly when either heating orcooling of the cantilever beam.

Suitable AOMs are the ATD-80 series AOM manufactured by IntraAction Corp(www.intraaction.com), or AOMs available from AA Opto-Electronic, seewww.aaoptoelectronic.com. The IntraAction device operates at 80 MHz, butcan be driven by modulating the frequency of the RF input signal toachieve beam scanning via Bragg deflection over a range of up to 40mrad. If this angular deflection is too small then it could be increasedusing a suitable lens amplification unit. It is also possible to achieve2D scanning by using two AOM devices in series.

FIG. 6c shows another way of independently controlling the amount ofphotothermal energy being delivered to each probe during a scan period.In this example, instead of illuminating each probe continuously betweenT₁ and T₂ and between T₃ and T₄ (as in FIG. 8b ) the beam is turned onand off quickly so that each probe is illuminated intermittently in aseries of pulses within each illumination period (or pulse train period)and the “mark-space ratio” of the pulses is varied to control the amountof energy delivered. So in this example during a single scan sequencethe first probe is illuminated by a pulse train 55 of eight relativelylong pulses and the second probe is illuminated by a pulse train 56 ofeight pulses which each have the same average intensity but a shorterduration than the pulses of the pulse train 55.

FIG. 6d shows yet another way of independently controlling the amount ofphotothermal energy being delivered to each probe during a single scanperiod. In this example, instead of illuminating each probe continuouslybetween T₁ and T₂ and between T₃ and T₄, the beam is turned on and offso the first probe is illuminated by a single relatively long pulse 57and the second probe is illuminated by a single pulse 58 which has thesame average intensity but a shorter duration than the pulse 57.

In yet a further embodiment shown in FIG. 7 the intensity of the inputbeam 53 and actuation beam 52 is kept constant and the amount ofphotothermal energy delivered to each probe during a single scan periodis controlled instead by controlling the angle of the actuation beam viathe AOM. FIG. 7 shows two scan sequences in which the angular sweep ofthe actuation beam is stopped as it reaches the first and second probesand held during Dwell Periods A-D. The lengths of the Dwell Periods A-Dcan then be controlled to independently control the amount ofphotothermal energy being delivered during a scan sequence. In thisexample Dwell Periods A-D are all different.

Precise timing of the pulses 55-58 in relation to the actuation beamposition is achieved by calibration. Such calibration is desirablebecause:

-   -   a. the photothermal energy input to a given cantilever is        controlled by timing the modulation of the actuation beam 52        with respect to the position of the actuation spot as it scans        over the array. However the exact position of the actuation beam        spot is unknown as it is a function of the dynamic response of        the AOM 51 and the detailed optical configuration; and    -   b. the actual motion of the probe in response to the        photothermal energy input will be filtered by the thermal time        constant of the probe itself.

Calibration can be achieved by using the probes themselves and theinterferometer based sensing system 10,12,14. A typical calibrationprocedure might be as follows.

The AOM 51 is initially driven at low frequency with a sawtooth waveformof the required amplitude. This sawtooth periodically deflects theactuation beam 52 across the probes and, in the absence of lasermodulation, the probes are addressed at the sawtooth frequency. Theprobes respond to the photothermal energy input by deflecting and thenreturning to zero deflection as the probe cools down, with someoscillation around zero due to the high quality factor. This initialprocedure gives a rough indication of the photothermal response, thethermal time constant, and the quality factor of each probe. Furtheradjustments can be made by moving the central position of the sawtoothscan pattern back and forth and observing the motion of the outercantilevers as the sawtooth scan moves off them. In this way the beamscan can be approximately centralised.

The frequency of the sawtooth waveform is now increased and the motionof the probes observed by the interferometer based sensing system. Anychange in the scan amplitude or scan centre can be adjusted by followingthe above procedure. The motion of the probes will transition from theclassic impulse response of a harmonic oscillator to a DC deflectionwith the scan modulation superposed upon it. As the scan frequencycontinues to increase up to the operating frequency, the address rate ofthe actuation beam exceeds the thermal relaxation time of the probes andthe probes stabilise at a steady DC deflection. Some variation indeflection between probes may be apparent at this stage and can be usedto calibrate their individual response functions, by increasing theactuation beam energy and noting the increase in deflection of eachprobe. However, because the position and corresponding dwell time of theIR laser is not a priori known, such a calibration may be subject to adegree of error.

It is now necessary to implement individual control of the probes. TheAOM drive signal 58 provides an indication of the position of theactuation beam at any point in time, which can be obtained by relatingthe timing of the sawtooth waveform to the previous calibration results.However a more direction measurement could be made by inserting a 45degree semi-reflecting mirror 81 into the beam path above the objectivelens to create a weak reference beam. The reference beam then passesthrough an equivalent lens 82 and is focused onto a photodiode 83 (orequivalent) that effectively parallels the operation of the probe arraybut with a much faster response. The signal from the photodiode 83 canbe used for timing purposes and may be more accurate than the AOM drivesignal 58. Alignment and calibration of the photodiode 83 can beundertaken at lower frequencies, alongside the probe array, as above.

Sequential scanning actuation can be implemented using a variety oftiming schemes but for this example the actuation scheme of FIG. 6c willbe referred to. In this scheme it is necessary to trigger a pulse train55, 56 of the required energy when the actuation beam is passing overeach probe. A timing signal can be obtained by using the signal from theIR photodiode 83, which will send out a pulse train as the actuationbeam passes over the probe array or alternatively by using the AOMsignal once calibrated by the IR photodiode. The timing signal is thenused to trigger the actuation pulse train 55, 56 for each probe. Anyprobe can be individually calibrated for a range of pulse trainenergies. Fine tuning of the position of the pulse train 55, 56 overeach probe can be achieved by varying the timing of the pulse train sothat it strays over one or the other side of the probe, which will beapparent from the reduction in the deflection of the probe as the IRenergy is lost. In this way the pulse can be centred on the probe toensure an accurate and reproducible photothermal actuation.

The actuation light source 50 and AOM 51 can be considered to providethe drive mechanism for both the z position feedback and the probeoscillation. That is, they are set to drive oscillation of eachcantilever probe and to adjust each probe—sample separation distanceduring the course of a scan. Specific embodiments of the imaging modesof the SPM will now be described in more detail.

The probe microscope is especially suitable for examining large,substantially flat, surface areas to detect features on the nanometerscale, at the high speeds required for effective industrial application.A prime example of such an application is the inspection of ExtremeUltra-Violet (EUV) masks. A critical issue to be addressed forcommercialization of EUV lithography is the detection and reduction ofdefects on the masks and in particular on blank mask. Typical defects onblank masks are either pits or particles which can originate either onthe substrate, during multilayer deposition or on top of the multilayerstack. The buried defects are especially problematic, and 10 nm defectsand small may be considered an issue. For example, the phase shiftcaused by a 3 nm variation in mask substrate flatness is sufficient toproduce a printable defect. SPM is in principle well suited to thedetection of such defects, but single probe instruments are too slow forindustrial applications. A probe instrument of typically ten probes ormore is required to achieve acceptable scan speeds. The exemplaryoperating mode of such an instrument, which is based on a cyclicoscillation of each probe, is described in the following section.

It may be desirable to only have a subset of the probes at an imagingposition at a set height above the surface. For example with an array often probes it may be desirable to only have five probes (for exampleevery other probe) at an imaging position at any one time. Then if oneof the five probes becomes damaged, the five probes can be retractedfrom the sample 1 a and the other five probes moved into position. Suchslow gross probe z positioning can be achieved by the laser 50 which canoperate to select or retract individual probes (or groups of probes)from the imaging position independently of the other probes in thearray.

Each probe is continually monitored throughout its oscillation by theparallel interferometric detection system 14, which outputs a signal 60for each probe that corresponds to the instantaneous position of theprobe at a given point in time. Considerable amounts of data aregenerated in this way for a high speed scanning system. The SPMcontroller 3 therefore incorporates a field programmable gate array(FPGA) which is configured in order to provide the necessary processingcapability. As is known in the art, alternative signal processingtechniques such as digital signal processing (DSP) or a dedicatedanalogue or digital electronic method may be used. The probe cyclicmotion typically has a frequency range of 10's to 100's of kHz andsampling frequency for data recording is in the region of 100 MHz.Consequently, each cycle of probe movement is sampled in the region of1000 to 10,000 times, which is more than sufficient to analyse theheight detector signal 60 to obtain a surface height detection point foreach tip in the array. There are a number of ways that the instantaneousheight detection signal can be processed to derive a surface heightreading for any given probe. However in the simplest case readings canbe based on the lowest recorded point in the probe oscillation cycle,when the probe tip is considered to be substantially in contact (orclose to contact) with the surface.

The xy scanner 2 translates the cantilever array across the surface ofthe sample in order to generate an image of the surface. The controller3 ensures that the tracking mirror 17 is matched with the scan patterndriven by the scanner 2 such that light from both the IR actuationsource 50 and the detection source 10 maintain their position on eachprobe (shown in FIG. 5) in the array as it moves. The controller 3 maycalculate different drive signals for the scanner 2 and tracking mirror17 depending on their particular construction and mechanical behaviour.If the sample is scanned the tracking mirror 17 may not be required ifthe sample is moved such that the probe, detection and actuation systemremain in a fixed registration.

In this way the probe array is scanned over the surface, with themicroscope collecting data from each probe within the array to provide aspatial map of the surface, formed of data points with sub-nanometervertical and horizontal resolution. It will be appreciated that manyscan operating patterns can be followed to collect data, depending onthe kind of inspection information required. In the case ofinvestigating defects on EUV masks, a large surface area must beinspected due to the low levels of defects. Typically the scan patternwill follow a raster pattern, with ten probes providing a dataacquisition rate increase of about ten times compared with the case of asingle probe microscope.

It will be appreciated that as the array is translated in the XY plane,each probe tip will encounter a different surface segment at the lowpoint of each probe oscillation cycle as it engages with the surface. Asthe surface is not completely smooth, the tip will therefore engage atdifferent points in the oscillation cycle and the surface height of agiven segment extracted as described above. No adjustments are made tothe cantilever base height on a segment by segment basis, as inconventional SPM where a feedback loop operates. However forsubstantially flat samples scanning the array at a constant separationwith the sample and not making any adjustments may be acceptable, andconfer significant advantages. For example, it should be emphasized thatthe scan speed of the parallel probe microscope is not limited by thez-actuation feedback loop when operated in this mode, as is the casewith a conventional SPM. The parallel probe microscope is capable ofoperating at scanning speeds considerably in excess of the limit imposedby feedback on reasonably flat samples. The height information extractedby the parallel interferometer detection system 14 represents the trueinstantaneous height of the probe, rather than the output of a Zactuator servo control loop as utilized in conventional SPM. However,the SPM controller 3 is used to maintain the probe array within asuitable range of the surface over longer time periods. This is achievedby processing the height data for each probe to extract a parameter thatis indicative, for example, of the dwell time of the probe tip on thesurface. Other parameters could be employed. These parameter values foreach probe are then processed and used to drive a relatively slow zactuation control loop which adjusts the laser 50, the sole purpose ofwhich is to maintain probe motion within set limits.

One mode of operation of the microscope of FIG. 1 is shown in FIGS. 8a-d. A first probe traverses a part of the sample a step 130 shown in FIG.8a . FIG. 8b shows the photothermal intensity being delivered to thefirst probe with respect to time over four cycles (each cycle having thesame approach/retract period P). FIG. 8c shows the output of theinterferometer for that probe—i.e. the height of the probe tip relativeto the stage 1. The interferometer analyses the height signal of FIG. 8cto provide an indication of the point in the cycle at which the probetip can be considered to have made contact with the sample or beproximate the sample.

During each cycle the probe starts retracted from the sample at heighth₁, then the photothermal intensity shown in FIG. 8b is increased so theprobe approaches the sample until it reaches a surface position atheight h₂ at which the probe interacts with the sample. In response todetection of the interaction with the sample the controller 3 reducesthe photothermal intensity which causes the probe to retract from thesample. After the probe has traversed the step, it can be seen that theprobe is retracted at an earlier point in the cycle.

At the same time as the first probe traverses the step 130 shown in FIG.8a , a second probe scans across another part of the sample and meets adifferent step. FIG. 8d shows the photothermal intensity being deliveredto the second probe with respect to time over the same time periodT_(start) to T_(finish). Note that the second probe meets a step earlierthan the first probe.

The microscope uses the height signal of FIG. 8c to form an image of thesample. The height signal may be used in a number of ways to form theimage. For example the image of the sample may comprise a plurality ofpixels, each pixel varying in accordance with the value of the heightsignal when the surface position is detected for a given single cycle ofthe motion of the probe towards and away from the sample. Alternativelyeach pixel of the image may be derived from plural height data samplescollected from the height signal over an extended portion of the probemotion during a single cycle of its motion, rather than from a singledata sample for each cycle. For example plural height data samples maybe collected for an extended portion of the probe motion before and/orafter the surface position is detected, and these samples analysed todetermine the value of a material property (such as elasticity) and thatmaterial property used as a pixel of the image. In this way an image ormap of the material property across the sample can be formed.

Further details of the imaging mode described above can be found inWO/2012/104625, the contents of which are incorporated herein bereference.

Optionally the microscope of FIG. 1 also has an optical system forgenerating an image of the beamlets on the probes, which will now bedescribed. An illumination source 62 generates an illumination beam 63which illuminates the probes via beam splitters 64, 65 (such as dichroicmirrors) and the objective lens 18. The beamsplitter 65 transmits thebeamlets 15 a-c but reflects the illumination beam 63 which hasdifferent wavelengths to the beamlets 15 a-c.

Reflected or scattered light from the probes is directed onto a chargecoupled device (CCD) camera 66 by a tube lens 67. The CCD camera 66generates an image of the complete array of probes (similar to FIG. 6)which is input to the controller 3. The image can then be used by thecontroller 3 to adjust the XY position of the array of probes and/or theangles of the beamlets from the SLMs 12,51 so that the beamlets arecentred on the probes prior to imaging. Once the beamlets have beencrudely centred on the probes using the image from the CCD camera 66,the controller 3 can then use the signals from the interferometerphotodiode 22 to adjust the XY position of the array of probes and/orthe angles of the beamlets from the SLMs 12,51 so that the beamlets areaccurately centred on the probes. This is done by moving the beamlet orthe probe to the left side of the probe until the beamlet falls off theleft edge of the probe (and the associated signal from the photodiodechanges abruptly or drops below a pre-set level); then moving thebeamlet or the probe to the right side of the probe until the beamletfalls off the right edge of the probe (and the associated signal fromthe photodiode changes abruptly or drops below a pre-set level). Thebeamlets or probe can then be moved to the midpoint between thesepoints. A similar process can be used to accurately position thebeamlets relative to the distal end of the cantilever.

This accurate xy alignment can be achieved by moving the probes inunison by moving the probe support 7, or more preferably byindependently adjusting the angles of the beamlets from the SLM 12.

A microcantilever biosensor will now be described with reference to FIG.9. Many of the components are equivalent to those shown in FIG. 1, andthe same reference numbers will be used for such equivalent components.However there is no requirement for probe array scanning, and the probearray is housed within a microfluidic cartridge 70 with an optical portthat is capable of delivering the fluid to be analysed to the probearray while allowing optical access. It will be appreciated that suchmicrofluidic systems are well known in the art and so will not bedescribed further here. Tracking mirror 17 from FIG. 1 is omitted andreplaced by an angled dichroic mirror 17 a.

The probe array could be comprised of silicon or silicon nitridecantilevers as described above, except that no tip is necessary, as theactive biosensor area of the cantilever is generally a well-defined areaon the cantilever back, rather than on the tip. An area on the back ofthe cantilever will therefore be coated with a bimetallic layer forphotothermal actuation, while another segment will typically be coatedwith gold for the biosensor, generally well away from both the actuationand detection lasers to avoid photodegradation. Gold is often usedbecause it is suitable for the immobilization of thiol modifiedbiochemical entities. These are the receptors which bind the targetmolecules (ligands) to the biosensor surface, thereby inducing surfacestress and increasing the mass of the probe overall. It will beappreciated that many receptor-ligand combinations exist and are wellknown in the art, and so will not be described in detail here. The exactnumber of probes in the array also varies depending on the biosensorapplication, because clinical applications often have multiple targetmolecules. Typically arrays might consist of 10's or 100's ofcantilevers, including both active and reference cantilevers. Referencecantilevers are typically used for temperature compensation duringmicrofluidic operation and analyte measurement.

The cantilever probe array and microfluidic system 70 is incorporatedinto the parallel interferometer and photothermal scanning actuationsystem in much the same way as the SPM of FIG. 1. The biosensor arraycan be operated in either static or dynamic modes as described above.The dynamic mode will be described below in more detail.

In the dynamic mode the increase of mass of each probe produces areduction in the resonant frequency that can be used to record thepresence and concentration of the ligand in the microfluidic cavitysurrounding the array. The parallel biosensor system is configured withthe probe array suitably functionalized and aligned within theinterferometer 14, with the cantilevers illuminated by the SLM 12 andAOM 51 to detect and actuate motion of the probes. In this case eachprobe is designed and fabricated to have the same resonant frequency,although some small variation is inevitable and not critical to thesystem operation. The analyte fluid is introduced into the microfluidicsystem 70 and is typically pumped to the probe array cavity foranalysis, or driven by capillary action.

There are a number of detection schemes that can be used to sense theresonant frequency shift that takes place as the ligands bind to thereceptors. In the case of amplitude detection, the probes are initiallydriven by the IR laser 50 at a frequency close to the unbound resonantfrequency of the probe array. In the case considered here the drivefrequency is below resonance, situated in a region of the resonant curvewhere the amplitude of probe oscillation varies approximately linearlywith frequency. For any given probe, the new probe resonant frequencybegins to drop as the ligand is bound, bringing the probe resonantfrequency closer to the photothermal drive frequency. As a consequencethe amplitude of the probe oscillation increases, approximately linearlywith the bound mass of the ligands. Alternative detection schemes basedon phase sensitive operation are known in the art and so will not bedescribed here.

Both amplitude and phase detection schemes are less effective when theprobes are heavily damped, as occurs when the probes are immersed incertain liquids, although they have the advantage of simplicity ofoperation as the same actuation drive signal can be used for all probes.However, sequential scanning allows individual control of each probe,thereby allowing improved detection schemes to be implemented. Forexample, in frequency modulation, the probe motion is fed back via abandpass filter and phase shifter to drive the actuation signal, therebycreating a self-exciting oscillator running at the resonant frequency ofthe probe. This scheme has greater robustness when operating in liquidenvironments. The band-pass filter is used to eliminate spuriousresonances that can occur. In order to implement the FM scheme withmultiple probes, the actuation beam is scanned sequentially by the AOMover each probe. The actuation beam completes the self-excited feedbackloop for each probe, ensuring the loop operates at the resonantfrequency of the probe, which changes as the ligands bind to thesurface. The output of each loop therefore corresponds to the shift inmass of the probe as a function of time. Other detection schemes, suchas those based on quality factor enhancement, are known in the art (forexample see U.S. Pat. No. 6,906,450) and could also be implanted withsequential scanning.

It will be appreciated that the simplified description given above couldbe amplified to take into account many of the more detailedconsiderations, such as compensation schemes and calibration, that arenecessary for successful biosensor operation. Such details fall outsidethe scope of this application and are well known in the art.

The detection and actuation scheme described above offer many advantagesfor biosensor operation. Clinical applications often require multipletargets and hence probes, making conventional PSD detection impractical.The inventive system could in principle be scaled to several 100's ofprobes, this covering many clinical diagnostic requirements. Thedetection and actuation system is also flexible, and can be reconfiguredby re-programming the controller 3 to operate with biosensor arrays ofdifferent dimensions and characteristics. The probe array itself issimple, cheap and disposable which is essential for many applications.The probe array, typically integrated into a microfluidic cartridge 70,can be introduced into the system quickly and the alignment completelyautomated as the actuation beam and sensing beamlets can be directedunder computer control until the required optimum detection andphotothermal actuation conditions are reached.

In the embodiments of the invention described above, a single objectivelens 18 is used to direct the sensing beamlets 15 a-c and the actuationbeam 52 onto the probes. However, the use of a single common objectivelens is not essential and FIG. 10 shows an embodiment in which differentobjective lenses 18, 80 are used for the sensing beamlets 15 a-c and theactuation beam 52. The embodiment of FIG. 10 has various elements incommon with the embodiment of FIG. 1, and the same reference numbers areused for these elements. In a further embodiment, the objective lens 80may be omitted, and the AOM 51 illuminates the probes directly, not viaan objective lens.

Although the invention has been described above with reference to one ormore preferred embodiments, it will be appreciated that various changesor modifications may be made without departing from the scope of theinvention as defined in the appended claims.

The invention claimed is:
 1. A method of actuating a plurality of probesin a scanning probe microscope system, the method comprising deliveringenergy to the probes so that the probes deform relative to a sample,wherein the energy is delivered to the probes by sequentiallyilluminating them with an actuation beam in a series of scan sequences,wherein two or more of the probes are illuminated by the actuation beamin each scan sequence; and controlling the actuation beam so thatdifferent amounts of energy are delivered to at least two of the probesby the actuation beam during at least one of the scan sequences.
 2. Themethod of claim 1 wherein the method comprises controlling the actuationbeam so that at least two of the probes are illuminated by the actuationbeam for a different amount of time during at least one of the scansequences.
 3. The method of claim 1 wherein the method comprisescontrolling the intensity of the actuation beam so that at least two ofthe probes are illuminated by the actuation beam with a differentintensity during at least one of the scan sequences.
 4. The method ofclaim 1 further comprising controlling the actuation beam over time sothat different amounts of energy are delivered to at least one of theprobes by the actuation beam during two or more scan sequences.
 5. Themethod of claim 1 wherein each probe has a thermal time constant whichis longer than each of the scan sequences.
 6. The method of claim 1wherein the probes are sequentially illuminated by directing an inputbeam into an optical device; transforming the input beam to generate theactuation beam which is output from the optical device at an outputangle; and operating the optical device to modulate the output angle ofthe actuation beam over time so that the actuation beam is sequentiallydirected onto the probes.
 7. The method of claim 6 wherein the opticaldevice comprises an acousto-optic or electro-optic modulator with adiffractive element which diffracts the input beam to generate theoutput beam, and wherein the output angle of the output beam ismodulated over time by applying an acoustic or electric signal to thediffractive element, the signal modulating a refractive index of thediffractive element.
 8. The method of claim 1 wherein the plurality ofprobes comprises ten or more probes.
 9. The method of claim 1 furthercomprising: a. directing a detection input beam into an optical device;b. transforming the detection input beam with the optical device into aplurality of output beamlets which are not parallel with each other; c.splitting each output beamlet into a sensing beamlet and an associatedreference beamlet; d. simultaneously directing each of the sensingbeamlets onto an associated one of the probes to generate a reflectedbeamlet; e. combining each reflected beamlet with its associatedreference beamlet to generate an interferogram; and f. measuring eachinterferogram to determine the position of an associated one of theprobes.
 10. The method of claim 1 wherein when each probe is illuminatedby the actuation beam it is heated and deforms to move the proberelative to a sample.
 11. The method of claim 1 wherein each probecomprises two or more materials with different thermal expansioncoefficients which are arranged such that when the probe is illuminatedby the actuation beam it is heated and deforms to move the proberelative to a sample.
 12. Actuation apparatus for actuating a pluralityof probes in a scanning probe microscope system, the apparatuscomprising: a beam source for generating an input beam; an opticaldevice arranged to receive the input beam and transform the input beamto generate an actuation beam which is output from the optical device atan output angle; and a controller arranged to operate the optical deviceto modulate the output angle of the actuation beam over time so that theactuation beam is sequentially directed onto the probes therebydelivering energy to the probes so that the probes deform relative to asample, wherein the energy is delivered to the probes by sequentiallyilluminating them with the actuation beam in a series of scan sequences,wherein two or more of the probes are illuminated by the actuation beamin each scan sequence; and the controller is arranged to operate theoptical device and/or the beam source and/or an optical element in apath of the actuation beam or in a path of the input beam so thatdifferent amounts of energy are delivered to at least two of the probesby the actuation beam during at least one of the scan sequences.
 13. Theapparatus of claim 12 wherein the optical device comprises anacousto-optic or electro-optic modulator with a diffractive elementwhich diffracts the input beam to generate the output beam, and whereinthe output angle of the output beam is modulated over time by applyingan acoustic or electric signal to the diffractive element, the signalmodulating a refractive index of the diffractive element.
 14. A probesystem comprising a plurality of probes; and actuation apparatusaccording to claim
 12. 15. A probe system comprising a plurality ofprobes; and actuation apparatus according to claim 13.